Somatic gene therapy to suppress secondary cataract formation following eye surgery

ABSTRACT

Disclosed is a replication-recombinant virus, preferably an adenovirus that lacks E1a, E1b and E4 ORF 6, capable of infecting an eye and comprising a lens epithelial cell specific promoter driving an ORF encoding at least one protein, which when expressed in lens epithelial cells of an eye suppresses, at the level of the germinative epithelium of the lens of the eye, cellular proliferation which is stimulated by eye surgery and which would otherwise result in secondary cataract formation in the eye. Also disclosed is the use of the recombinant virus for the treatment of an eye, undergoing eye (e.g. cataract) surgery, in order to reduce the incidence of cellular proliferation in the eye following the surgery and thereby to prevent the formation of secondary cataracts.

RELATED APPLICATION

[0001] This application is a continuation of U.S. Ser. No. 09/710,035,filed Mar. 10, 2000, which is a continuation application of U.S. Ser.No. 08/867,902, filed Jun. 3, 1997, which was issued as patent numberU.S. Pat. No. 6,200,799 B1 on Mar. 13, 2001, each of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] This invention relates to materials and methods for theconstruction of recombinant viral, particularly adenoviral, deliverysystems that provide lens epithelial cell type specific regulation ofexpression of proteins which inhibit cellular proliferation. Thisinvention also relates to the use of these recombinant viral deliverysystems for inhibiting or preventing the formation of secondarycataracts following surgical intervention on the eye.

[0003] Cataract operations are the second most frequent operation in thewestern world. The cataract is an affliction characterized by anopacification of the crystalline lens of the eye which reduces thevisual acuity of the patient. The surgical technique currently utilizedfor removing cataracts consists of removing the crystalline with a smallaspirator. Access to the interior of the crystalline capsule is achievedby a circular opening at the anterior face of the lens capsule. Thispermits the surgeon to insert a new lens, which will restore the eye'sability to focus incoming light on the surface of the retina.

[0004] Secondary cataracts constitute the most frequent complicationfollowing this operation, occurring in up to 50% of adult eyes and evenmore frequently following surgery of children with congenital cataracts(Nischi et al., 1986). (Surgical interventions on the eyes of rabbitsalso result in secondary cataract formation (100%) in the weeksfollowing the operation (Blomstedt. et al., 1987)). Human secondarycataracts are characterized by a secondary opacification of theposterior capsule appearing in the months following surgery. They arethe consequence of the stimulated proliferation at the level of thegerminative epithelium of the lens, which are the only cells withdivision potential in the mature eye. Laser treatment, which is theaccepted means of treating secondary cataracts, unfortunately risksadditional complications, the most notable of which is detachment of theretina (Salvenson et al., 1991).

SUMMARY OF THE INVENTION

[0005] In accordance with this invention is provided areplication-defective recombinant virus that can infect an eye of apatient and that contains:

[0006] an Open Reading Frame (ORF) which, when expressed in lensepithelial cells of the eye, suppresses, at the level of the germinativeepithelium of the lens of the eye, cellular proliferation which isstimulated by eye surgery and which would otherwise result in secondarycataract formation in the eye;

[0007] the ORF, to be expressed, being under the control of a promotersequence which is active exclusively in human lens epithelial cells.

[0008] The preferred replication-defective recombinant virus is areplication-defective recombinant adenovirus, especially an adenoviruslacking E1a, E1b and E4 ORF 6.

[0009] The preferred ORF, to be expressed, is one of the following:

[0010] a) an ORF encoding a protein that regulates the cell cycle,especially a non-phosphorylatable retinoblastoma (Rb) gene;

[0011] b) a dominant negative mutant of an ORF encoding a proteininvolved in signal transduction, especially a dominant negative mutantof a RAS gene, particularly the codon 116 mutant of the human RAS gene;or

[0012] c) an ORF encoding a protein which will severely disrupt DNAreplication in human lens epithelial cells, especially a thymidinekinase gene of a Herpes virus, particularly the Herpes Simplex type 1thymidine kinase (HSTK) gene

[0013] (to be used in conjunction with a treatment of the patient withacyclovir or a nucleoside analogue thereof).

[0014] The preferred promoter sequence is one of the following:

[0015] a) a promoter of a human Major Intrinsic Protein (MIP) gene,preferably the portions of the MIP gene from −259 nt to +34 nt, or

[0016] b) a promoter of a human β A3/A1-crystallin gene, preferably theportions of the β-crystallin gene from −345 nt to +45 nt, or preferably

[0017] c) a composite promoter comprising either the MIP promoter or theβ-crystallin promoter or portions thereof, in combination with elementsof an early growth response gene promoter such as the rat Early GrowthResponse-1 (EGR-1) gene promoter from −518 nt to −236 nt of the EGR-1gene.

[0018] Also in accordance with this invention are provided:

[0019] a) purified and isolated DNA sequences of the composite EGR-1/MIPand composite EGR-1/β-crystallin promoters and

[0020] b) vectors for the formation of replication-defective recombinantviruses containing the gene, to be expressed, under the control of thepromoter sequence which is expressed exclusively in human lensepithelial cells.

[0021] Further in accordance with this invention is provided a methodfor the treatment of an eye, undergoing eye (e.g. cataract) surgery, inorder to reduce the incidence of cellular proliferation in the eyefollowing the eye surgery, and thereby prevent the formation ofsecondary cataracts, comprising the step of:

[0022] treating the eye with the replication-defective recombinantvirus, particularly adenovirus, of this invention, preferably during theeye surgery.

BRIEF DESCRIPTION OF THE FIGURES

[0023]FIG. 1 shows schematically the two-step process for theconstruction of a replication-defective recombinant adenovirus inaccordance with this invention.

[0024]FIG. 2 is a restriction map of plasmid pad5ΔE4ORF6, used in theprocess shown in FIG. 1 and described in Example 8.

[0025]FIG. 3 is a restriction map of the plasmid pXC 15-18 used in theprocess shown in FIG. 1 and described in Example 8.

[0026]FIG. 4 is the DNA sequence of the rat EGR-1 promoter (SEQ ID NO:1)of Example 1.

[0027]FIG. 4A is a restriction map of plasmid, pxcEGR, of Example 1.

[0028]FIG. 5 is a restriction map of the GFP ORF of Example 1.

[0029]FIG. 5A is a restriction map of plasmid pxcEGR-GFP of Example 1.

[0030]FIG. 6 is a restriction map of the dominant negative human RAS ORFof Example 1.

[0031]FIG. 6A is a restriction map of plasmid, pxcEGR-RAS, of Example 1.

[0032]FIG. 7 is a restriction map of the Rb ORF of Example 1.

[0033]FIG. 7A is a restriction map of plasmid, pxcEGR-Rb, of Example 1.

[0034]FIG. 8 is a restriction map of the HSTK ORF of Example 1.

[0035]FIG. 8A is a restriction map of plasmid, pxcEGR-HSTK, of Example1.

[0036]FIG. 9 is the DNA sequence of the human MIP promoter (SEQ ID NO:2)of Example 2.

[0037]FIG. 9A is a restriction map of plasmid, pxcMIP, of Example 2.

[0038]FIG. 10 is a restriction map of plasmid, pxcMIP-GFP, of Example 2.

[0039]FIG. 11 is a restriction map of plasmid, pxcMIP-RAS, of Example 2.

[0040]FIG. 12 is a restriction map of plasmid, pxcMIP-Rb, of Example 2.

[0041]FIG. 13 is a restriction map of plasmid, pxcMIP-HSTK, of Example2.

[0042]FIG. 14 is a restriction map of plasmid, pxcComp1, of Example 3.

[0043]FIG. 14A is the DNA sequence (SEQ ID NO:3) of the composite 1promoter of pxcComp1 of Example 3.

[0044]FIG. 15 is a restriction map of plasmid, pxcComp1-GFP, of Example3.

[0045]FIG. 16 is a restriction map of plasmid, pxcComp1-RAS, of Example3.

[0046]FIG. 17 is a restriction map of plasmid, pxcComp1-Rb, of Example3.

[0047]FIG. 18 is a restriction map of plasmid, pxcComp1-HSTK, of Example3.

[0048]FIG. 19 is the DNA sequence of the human β-crystallin promoter(SEQ ID NO:4) of Example 4.

[0049]FIG. 19A is a restriction map of the plasmid, pxcβCrys1, ofExample 4.

[0050]FIG. 20 is a restriction map of plasmid, pxcComp2, of Example 5.

[0051]FIG. 20A is the DNA sequence (SEQ ID NO:5) of the composite 2promoter of pxcComp2 of Example 5.

[0052]FIG. 21 is a restriction map of the plasmid, pMT-E4 ORF6, ofExample 7.

DETAILED DESCRIPTION OF THE INVENTION

[0053] This invention involves the use of a replication-defectiverecombinant virus, preferably an adenovirus, to reduce the incidence ofcellular proliferation following surgical intervention on the eye ofmammals, especially humans. In particular, replicative-defectiverecombinant adenoviral vectors are used to deliver ORF whose encodedproteins suppress, at the level of the germinative epithelium of thelens of the eye, cellular proliferation that is stimulated by the eyesurgery and that would otherwise result in secondary cataract formation.

[0054] The particularly preferred replication-defective recombinantvirus is a recombinant adenovirus lacking E1a, E1b and E4 ORF 6. Howeverequivalents of this virus can also be used, such as HIV-derivedreplication-defective retroviruses and Adeno-associated viruses (Kearnset al., 1996; Liu, M L. et al., 1996; Russ et al., 1996; Xiao andSamulski, 1996), as well as non-viral equivalents such as liposomes(Hangai et al., 1996).

[0055] The preferred ORF, to be expressed, is one of the following:

[0056] a) an ORF encoding a protein that regulates the cell cycle,especially a non-phosphorylatable retinoblastoma (Rb) gene;

[0057] b) a dominant negative mutant of an ORF encoding a proteininvolved in signal transduction, especially a dominant negative mutantof a human RAS gene (Kaplan, 1994; Sung et al., 1996), particularly thecodon 116 mutant of the human RAS ORF; other examples of such ORFs arethe p16 and p21 genes (Kamb et al., 1994; Wu et al., 1996); or

[0058] c) an ORF encoding a protein which will severely disrupt DNAreplication in human lens epithelial cells, especially a thymidinekinase gene of the Herpes virus family, such as the Herpes Simplex type1 virus or Varicella zoster, or the UL-97 gene of Cytomegalovirus whichencodes a protein kinase (to be used in conjunction with a treatment ofthe patient with acyclovir or a nucleoside analogue thereof).

[0059] The ORF, to be expressed, is placed under the control of atissue-specific promoter so that the expression of the ORF occursuniquely in lens epithelial cells. In this regard, precise expression ofthe desired ORF in lens epithelial cells is preferably achieved by theuse of promoter elements of the human MIP gene (particularly from −259nt (nucleotide) to +34 nt), promoter elements of the humanbeta-crystallin gene (particularly from −345 nt to +45 nt) or promoterelements of both genes. Other lens epithelial cell-specific promoter canalso be used, such as the promoters of the human alpha, gamma, delta orepsilon-crystallin genes (Chepelinsky et al.,1991; Piatigorsky, 1989;Kondoh et al., 1988).

[0060] The promoters of the endogenous MIP and β-crystallin genes, likethe other lens epithelial cell specific promoters, are methylated in allcell types except differentiated lens epithelial cells. This methylationrenders the promoters inactive. The divisional event that precedesdifferentiation is accompanied by a de-methylation of the lens cellspecific promoters. It has been demonstrated that non-methylated lensspecific promoters are active in the germinal cells (Peek et al., 1991).Recombinant adenoviruses of this invention are therefore produced in thepresence of 5-aza-2′-deocytidine to ensure that no methylation of thelens specific promoters takes place during production of the recombinantvirus.

[0061] It is particularly preferred that rapid ORF expression, followingsurgery, be achieved by the presence of elements of the promoter of anearly growth response gene or an “immediate early gene” (Kiessling andGass, 1993; Weichselbaum et al., 1994), preferably cis rat EGR-1elements, in composite promoter constructs of this invention which alsocontain portions of the β-crystallin or MIP promoter. Such compositepromoter constructs of this invention are provided with:

[0062] a) first elements intended to provide rapid expression of theORFs, to be expressed, upon mitotic signaling, with subsequent decay ofexpression of the ORFs; these first elements are not believed to exhibittissue specificity; and

[0063] b) second elements comprising the promoter of the MIP gene and/orthe promoter of the β-crystallin gene; these second elements ensureexquisite tissue specific expression yet are less rapidly induced topromote expression following entry into lens epithelial cells.

[0064] The construction of a replicative-defective recombinantadenovirus in accordance with this invention takes place in two steps asschematically shown in FIG. 1.

[0065] In the first step, the ORF, to be expressed, is cloned downstreamof a particular promoter and of an intron and upstream of the poly Asite into the polylinker of a pXC plasmid (FIG. 3) which contains theleft end of adenovirus (nucleotides 1-5788 with a 2700 nt deletion ofthe E1a-E1b region between nucleotide positions 358 and 3328 in GenBankAccession No. M73260).

[0066] The resulting pXC plasmid (recombinant 1) is then co-transfectedwith a modified pJM17 plasmid, shown in FIG. 2, into a modified 293 cellline (Graham et al., 1977), which is a transformed human cell line thatconstitutively expresses Ad5 E1 proteins and can be induced tosynthesize the E4 ORF 6 protein. Due to homologous recombination betweenthe pXC plasmid with its adenovirus left-end construct, the pad5ΔE4ORF6plasmid, and the helper (complementing) function of the E1 and E4 ORF 6proteins provided by the 293/MTE4ORF6 cells, a replication-defectiverecombinant adenovirus is produced and packaged. The packagedrecombinant adenovirus produced by the transfected 293/MTE4ORF6 cellshas a deleted E-1a-E1b region that is replaced by the promoter and ORF,of interest, initially inserted into the recombinant 1 plasmid. Plaquepurifications (three times) and analysis of resulting recombinant virusare carried out to verify the sequence of the resulting recombinantvirus. The recombinant adenovirus is incapable of replicating in cellsother than modified 293 cells and thus constitutes simply a deliverysystem for the protein encoded by the ORF, to be expressed.

[0067] A rabbit model system, exhibiting essentially 100% secondarycataract formation in the 2-3 weeks following surgical intervention ofthe eye, can be used, as described below, to evaluate the efficiency ofsuppression of lens epithelial cell proliferation by the recombinantadenovirus vectors of this invention.

[0068] The following three ORFs are independently expressed inreplication-defective recombinant adenoviral vectors:

[0069] 1. a non-phosphorylatable Rb ORF

[0070] 2. a dominant negative mutant (at amino acid position 116) of thehuman RAS ORF and

[0071] 3. a Herpes Simplex type 1 thymidine kinase ORF.

[0072] Control of expression in the recombinant adenoviral vectors iswith one of the following four promoter sequences:

[0073] 1. a human Major Intrinsic Protein (MIP) gene promoter,

[0074] 2. a first composite promoter containing elements of the ratEarly Growth Response-1 (EGR-1) gene promoter (from −518 nt to −236 nt)and the human Major Intrinsic Protein (MIP) gene promoter (from −259 ntto +34 nt)

[0075] 3. a human β A3/A1-crystallin promoter (−345 nt to +45 nt) and

[0076] 4. a second composite promoter containing elements of the EGR-1promoter (from −518 nt to −236 nt) and the β-crystallin promoter (−345nt to +45 nt).

[0077] Correct expression of recombinant adenoviruses containing lensepithelial cell specific promoters is verified in in vitro cultures oflens capsular sacs of pig eyes which have been operated upon. Thegerminal epithelial cells of pig eyes capsular sacs, that have undergonecataract surgery, proliferate in a manner analogous to secondarycataract formation (Liu, C. et al., 1996; Wormstone et al., 1997).

[0078] A replication-defective recombinant adenovirus for eachpromoter-ORF construct is made by co-transfection of first stage vectorswith the modified pJM17 plasmid lacking E4 ORF 6 into the modified 293cell line with metal inducible expression of E4 ORF 6 protein.

[0079] In the next step, rabbit lens capsular sacs are infected at thetime of surgical intervention with an adenovirus transformed with thecoding sequence for the Green Fluorescent Protein (GFP), in order todetermine the efficiency and specificity of ORF delivery and expressionin accordance with the invention at different multiplicities ofinfection (MOIs). Infection of the eyes during surgery can be carriedout in a conventional manner in accordance with this invention, butpreferably, the eyes are infected by introducing the recombinantadenoviral vector at the final stage of the surgery, that is when thechamber is refilled with sterile physiological solution. Onceappropriate MOIs have been determined, the duration of foreign ORFexpression is evaluated. Subsequently, the efficiency of suppression ofcellular proliferation is analyzed in operated eyes, using therecombinant adenoviruses containing either the non-phosphorylatable RbORF, the dominant negative RAS ORF or the HSTK ORF (in conjunction witha treatment with acyclovir or a nucleoside analogue thereof).

[0080] Green Fluorescent Protein (GFP) is an auto-fluorescing proteinexpressed in jelly fish. The advantage of this protein is that itsexpression can be easily detected without the addition of any othercomponents, such as is necessary for many other marker ORF systems. Asource of the GFP ORF (FIG. 5) for the recombinant adenovirus constructof this invention is the pxcEGR-GFP plasmid shown in FIG. 5A. Theprotein encoding sequence is modified in two respects: i) the codonusage is modified to be translated optimally in mammalian cells and ii)the wavelength necessary to excite the protein to fluoresce is modifiedto be in the near visible, as opposed to the far ultraviolet, as is thecase for the native jellyfish protein.

[0081] A retinoblastoma (Rb) cDNA clone is obtainable from Dr. Hamel inthe department of Medical Genetics at the University of Toronto (Hamelet al., 1992). The clone contains eight point mutations at both serineand threonine residues, thus rendering the encoded retinoblastomaprotein non-phosphorylatable. (See FIG. 7.) Over-expression of this Rbprotein is known to result in cell cycle arrest in the Gi phase of thecell cycle (Hamel et al., 1992; Chang et al., 1995).

[0082] A Harvey RAS ORF construct (FIG. 6), containing a dominantnegative mutation at codon 116 (tyrosine replacing the wild-typeasparagine at amino acid position 116), is obtainable from Dr. Kuzumakiof Hokkaido University, Japan.

[0083] An ORF encoding Herpes Simplex type 1 thymidine kinase (HSTK), asshown in FIG. 8, is known from Waldman et al. (1983).

[0084] An evaluation of a stimulated immune response directed againstadenovirus in infected rabbits is undertaken to determine whether it ispossible to re-infect with recombinant virus without neutralization bythe immune system. In principle, the interior of the eye should be“protected” from the immune system, and multiple infections withreplication-defective recombinant adenoviral vectors should rendersuppression of lens epithelial cell proliferation more efficient than asingle infection at the time of the operation.

[0085] The surgical technique used on the rabbits is identical to thatused in the human cataract operation described above. This animal systemthus constitutes an ideal model to demonstrate the effectiveness ofsomatic gene therapy in suppressing post-operative cellularproliferation, causing secondary cataract. Any immune reaction,engendered by infection with a recombinant adenoviral vector, is easilyassessed by analysing “infected” rabbit sera for stimulated response toadenovirus capsid proteins. In the absence of an immune reaction, therelative efficiency of multiple infections of replication-defectiverecombinant adenovirus vectors is then evaluated.

[0086] When the recombinant adenovirus of this invention contains athymidine kinase ORF of a Herpes virus, particularly the Herpes Simplextype 1 thymidine kinase (HSTK) ORF, the patient is also treated withacyclovir or a nucleoside analogue structurally related to acyclovir,such as gancyclovir, famcyclovir and valacyclovir, preferably withacyclovir (Clive et al., 1983). Acyclovir and its nucleoside analoguesare phosphorylated by the Herpes thymidine kinase and subsequentlyincorporated into host cell DNA. Once incorporated, they function aschain terminating nucleotides, and thus, cells expressing the viralthymidine kinase and incorporating the phosphorylated analogue arespecifically targeted. There is no reported toxicity of acyclovir at theconcentrations appropriate for this use, and it can be given orally orby eye drops to the patient before, during or after surgery.

[0087] The Examples which follow illustrate the invention.

EXAMPLE 1

[0088] Construction of an Adenovirus First Stage Vector (Recombinant 1)Containing Elements of the Rat EGR-1 (Early Growth Response) GenePromoter.

[0089] Amplification of the rat EGR-1 gene promoter (shown in FIG. 3) isaccomplished by PCR. A 5′ oligonucleotide corresponding to −538 nt to−518 nt of the transcription start site containing a synthetic Bgl IIsite is used in conjunction with a 3′ oligonucleotide complementary to+136 nt to +156 nt with a synthetic Sac I site. The conditions of PCRare: 20 picomoles of each primer, 50 μM dNTPs, 200 ng rat DNA, 4%formamide (recrystalised), 1.5 mM MgCl in 100 μl. The initial cycle is97° for 2 ½ minutes, 72° for 5 minutes (add enzyme) 35 cycles of 95°C.-30 seconds, 63° C.-40 seconds, 72° C. 60 seconds. 5′ primer (SEQ IDNO:6)     Bgl II GA AGATCT AGC CTC AGC TCT ACG CGC CT                                  EGR 3′-primer (SEQ ID NO:7)     Sac IGAA GAGCTC ACA CTG CGG GGA GTG TAG GT                                   EGR

[0090] The resulting 674 nt product is cleaved with Bgl II and Sac I andcloned into the Bgl II-Sac I sites of PSL 1180 (Pharmacia), and M13forward and reverse primers are used to verify the sequence of thecloned rat EGR-1 promoter. The Bgl II-Sac I fragment of the resultingplasmid, pSL 1180-EGR, is then excised and cloned into the Bgl II-Sac Isites of a CMV promoter deleted pCiNeo vector (Promega). This cloning isverified by digestion with Sma I and Nru I. The resulting plasmid,pEGRiNeo, is then cleaved with Bgl II and Fsp I. The resulting 1435 ntfragment, containing the rat EGR-1 promoter, an intron, a multicloningsite and a polyA addition site, is then cloned into the Bam HI-Hpa Isites of pXC15-18 (FIG. 3; Schaak, personal communication). Theresulting clone, pxcEGR, is verified by digestion with numerousrestriction enzymes. A restriction map of pxcEGR is given in FIG. 4.

[0091] The following four protein coding sequences are cloned into thepxcEGR vector to form different recombinant 1 adenoviral vectors: GreenFluorescent Protein (GFP), dominant negative RAS, a non-phosphorylatableretinoblastoma (Rb) and a Herpes Simplex type 1 thymidine kinase (HSTK)ORF. Described below are the steps by which the appropriate cloning isaccomplished.

[0092] GFP:

[0093] A 716 nt Nhe I-Sal I fragment encoding the Green FluorescentProtein (GFP) is isolated from peGFP-Cl (Clontech) and cloned into theNhe I, Sal I sites of pxcEGR. Sequencing of the cloning junctions and ofthe GFP DNA insert is accomplished with T3 and T7 primers. FIG. 5 is arestriction map of the GFP ORF, and FIG. 5A is a restriction map of theresulting plasmid, pxcEGR-GFP.

[0094] Dominant Negative RAS:

[0095] A Sac II (blunted by a Klenow fill-in reaction)-Xba I fragment ofdominant negative ras cDNA, obtained from Dr. Kuzumaki, is cloned intothe Nhe I (blunted by a Klenow fill-in reaction) -Xba I sites of pxcEGR.The correct clone is initially verified by restriction enzyme mapping.Sequencing of the cloning junctions and of the ras cDNA insert areperformed with T3 and T7 primers. FIG. 6 is a restriction map of thedominant negative RAS ORF, and FIG. 6A is a restriction map of theresulting plasmid, pxcEGR-RAS.

[0096] Rb:

[0097] A 2768 nt Eco RI-Sau I (Klenow fill-in blunted) fragment frompECE34 is ligated into the Eco RI-Not I (Klenow fill-in blunted) sitesof pxcEGR. The correct clone is initially identified by Sac I digestion.Sequencing of the cloning junctions and of the non-phosphorylatable RbcDNA insert are performed with T3 and T7 primers. FIG. 7 is arestriction map of the non-phosphorylated Rb ORF, and FIG. 7A is arestriction map of the resulting plasmid, pxcEGR-Rb.

[0098] HSTK:

[0099] A 1176 nt fragment of the Herpes Simplex type 1 thymidine kinaseORF is amplified by PCR using primers containing synthetic restrictionenzyme sites, a Eco RI at the 5′ end and a Hind III site at the 3′ end.The ORF is amplified by PCR, using the following two primers: 5′-primerCTGAATTCCTTGTAGAAGCGCGTATGGC (SEQ ID NO:8) 3′-primerCGCAAGCTTCTCCTTCCGTGTTTCAGTT (SEQ ID NO:9)

[0100] The thermal profile is 94° C., 5′; 58° C. 2′30; 72° C., 2′: 1 x;then 94° C., 30″; 58° C., 1′30″; 72° C., 2′: 30 x; then 72° C., 6′: 1 x.Standard PCR buffer conditions are employed. The amplified DNA fragmentis cloned into the Eco RI-Hind III sites of BlueScript vector(Stratagene). The sequence is verified with T3 and T7 primers. Thisfragment is cut with Hind III, rendered blunt by a Klenow fill-inreaction, and digested with Eco RI. This fragment is then cloned intothe Eco RI-Not I (blunted) sites of pxcEGR. FIG. 8 is a restriction mapof the Herpes Simplex type 1 thymidine kinase ORF, and FIG. 8A is arestriction map of the resulting plasmid, (pxcEGR-HSTK.

EXAMPLE 2

[0101] Construction of an Adenovirus First Stage Vector (Recombinant 1)Containing Elements of the MIP (Major Intrinsic Protein) Gene Promoter.

[0102] Amplification of the human MIP gene promoter (shown in FIG. 9) isaccomplished by PCR. A 5′ oligonucleotide corresponding to −259 nt to−239 nt of the transcription start site containing a synthetic Bgl IIsite at its 5′ end is used in conjunction with a 3′ oligonucleotidecomplementary to +14 nt to +34 nt with a synthetic Sac I site. Theconditions of PCR are: MIP PCR, one cycle with a 63° C. annealing andthen a two step cycle of 94° and 70° C. for 30 cycles. Standard PCRbuffer conditions are employed. 5′-primer (SEQ ID NO:10)    Bgl II GAAGATCT CTT CCA GTC CTG CTG TTC TT                                 MIP3′-primer (SEQ ID NO:11)     Sac I GAA GAGCTC ATG GTC ACA GTG CCT GGG TC                                 MIP

[0103] The resulting 293 nt product is cleaved with Bgl II and Sac I andcloned into the Bgl II-Sac I sites of PSL 1180 (Pharmacia), and M13forward and reverse primer are used to verify the sequence of the humanMIP promoter. The Bgl II-Sac I fragment of the resulting plasmid, pSL1180-MIP, is then excised and cloned into the Bgl II-Sac I sites of aCMV promoter deleted pCiNeo vector (Promega). This cloning is verifiedby digestion with Alw I. The resulting plasmid, pMIPiNeo, is thencleaved with Bgl II and Fsp I. The resulting 1435 nt fragment is thencloned into the Bam HI-Hpa I sites of pXC15-18 (FIG. 3). The resultingclone, pxcMIP, is verified by digestion with numerous restrictionenzymes. A restriction map of this clone is given in FIG. 9A.

[0104] As described above for the pxcEGR clones of Example 1, four ORFs(encoding GFP, dominant negative RAS, non-phosphorylatable RB and HerpesSimplex type 1 thymidine kinase) are cloned into pxcMIP to formdifferent recombinant 1 adenoviral vectors. The necessary enzymaticdigestions for the pxcMIP clones and the sequencing of the junctions andORF inserts of these clones are as described above for the pxcEGRclones.

[0105] GFP:

[0106] A 716 nt Nhe I-Sal I fragment containing the Enhanced GreenFluorescent Protein (GFP) is isolated from peGFP-C1 (Clontech) andcloned into the Nhe I-Sal I sites of pxcMIP. See restriction map ofresulting plasmid, pxcMIP-GFP, in FIG. 10.

[0107] Dominant Negative RAS:

[0108] The Sac II (blunted)-Xba I fragment of dominant negative RAS cDNAis cloned into the Eco RI (blunted)-Xba I sites of pxcMIP. The correctclone is initially verified by restriction enzyme mapping. Sequencing ofthe junctions and the RAS cDNA insert are performed with T3 and T7primers (See Map). See restriction map of resulting plasmid, pxcMIP-RAS,in FIG. 11.

[0109] Rb:

[0110] A 2768 nt Eco RI-Sau I (Klenow fill-in blunted) fragment frompECE34 is ligated into the Eco RI-Not I (Klenow fill-in blunted) sitesof pxcMIP. The correct clone is initially identified by Sac I digestion.See restriction map of resulting plasmid, pxcMIP-Rb, in FIG. 12.

[0111] HSTK:

[0112] A 1176 nt fragment of the Herpes Simplex type 1 thymidine kinaseORF is cut with Hind III (rendered blunt by a Klenow fill-in reaction)and Eco RI. This fragment is then cloned into the Eco RI-Not I (blunted)site of pxcEGR. Sac I digestion is initially used to verify this clone.See restriction map of resulting plasmid, pxcMIP-HSTK, in FIG. 13.

EXAMPLE 3

[0113] Construction of an Adenovirus First Stage Vector (Recombinant 1)Containing a Composite 1 Promoter with Elements from both the Rat EGR-1(Early Growth Response) Gene and Major Intrinsic Protein (MIP) GenePromoter Elements.

[0114] Plasmid pSL1180-MIP of Example 2 is cleaved with Bgl II, followedby a Klenow fill-in reaction. Both enzymes are heat inactivated, andthen the plasmid is further digested with Sac I. The resulting 293 ntsfragment is isolated from an agarose gel.

[0115] Plasmid pSL1180-EGR of Example 1 is digested with Sma I and BglII, and the resulting 282 nt fragment is isolated.

[0116] The two DNA fragments are then ligated overnight at roomtemperature. A small aliquot (1/100 of a microliter) is then used as atemplate in a PCR, using the 5′ EGR oligonucleotide of Example 1 and the3′ MIP oligonucleotide of Example 2. The resulting 585 nt compositepromoter fragment is digested with Bgl II and Sac I and isolated from anagarose gel. This fragment is then ligated into the Bgl II Sac I sitesof PSL 1180. Restriction enzyme digestion is used to initially confirmthe cloned PCR product. After confirmation by sequencing of thecomposite promoter, the Bgl II-Sac I fragment is isolated and ligatedinto the Bgl II-Sac I sites of Bgl II-Sac I digested pCiNeo, thusreplacing the CMV promoter with the composite promoter. This plasmidpCompiNeo is then digested with Bgl II and Fsp I. The resulting 1318 ntfragment is then ligated into the Bam HI-Hpa I sites of pXC15-18 (FIG.3), resulting in the plasmid pxcComp1. A restriction map of pxcComp1 isgiven in FIG. 14, and the DNA sequence of its composite 1 promoter isshown in FIG. 14A.

[0117] As described above for the pxcEGR clones of Example 1, four ORFs(encoding GFP, dominant negative RAS, non-phosphorylatable RB and HerpesSimplex type 1 thymidine kinase) are cloned into pXComp1 to formdifferent recombinant 1 adenoviral vectors. The necessary enzymaticdigestions for the pXComp1 clones and the sequencing of the junctionsand ORF inserts of these clones are as described above for the pxcEGRclones.

[0118] GFP:

[0119] A 716 nt Nhe I-Sal I fragment containing the Enhanced GreeenFluorescent Protein (GFP) is isolated from peGFP-Cl (Clontech) andcloned into the Nhe I-Sal I sites of pxComp1. See restriction map ofresulting plasmid, pxcComp1-GFP, in FIG. 15.

[0120] Dominant Negative RAS:

[0121] The Sac II (blunted)-Xba I fragment of dominant negative RAS cDNAof Example 1 is cloned into the Eco RI (blunted)-Xba I sites ofpxcComp1. The correct clone is initially verified by restriction enzymemapping. Sequencing of the junctions and the ras cDNA insert areperformed with T3 and T7 primers. See restriction map of resultingplasmid, pxcComp1-RAS, in FIG. 16.

[0122] Rb:

[0123] A 2768 nt Eco RI-Sau I (Klenow fill-in blunted) fragment frompECE34 is ligated into the Eco RI-Not I (Klenow fill-in blunted) sitesof pxcomp1. The correct clone is initially identified by Sac Idigestion. See restriction map of resulting plasmid, pxcComp1-Rb, inFIG. 17.

[0124] HSTK:

[0125] A 1176 nt fragment of the Herpes Simplex type 1 thymidine kinaseORF is cut with Hind III (rendered blunt by a Klenow fill-in reaction)and Eco RI. This fragment is then cloned into the Eco RI-Not I (blunted)site of pxcCompl. Sac I digestion is initially used to verify thisclone. See restriction map of resulting plasmid, pxcComp1-HSTK, in FIG.18.

Example 4

[0126] Construction of an Adenovirus First Stage Vector (Recombinant 1)Containing the Human β-crystallin A3/A1 Gene Promoter.

[0127] Amplification of the human β-crystallin A 3/A1 gene promoter(shown in FIG. 19) is accomplished by PCR. A 5′ oligonucleotidecorresponding to −330 nt to −311 nt of the transcription start sitecontaining a synthetic Bgl II site at its 5′ end is used in conjunctionwith a 3′ oligonucleotide complementary to +26 nt to +45 nt with asynthetic Sac I site. 5′ primer    Bgl II GAAGA TCTCCCAGGGTCTTAAGGT (SEQID NO:12) 3′ primer     Sac I GAAGAGCTCT TACTCACCCAGCTCCTGC (SEQ IDNO:13)

[0128] The resulting 375 nt product is cleaved with Bgl II and Sac I andcloned into the Bgl II-Sac I sites of PSL 1180 (Pharmacia), and M13forward and reverse primer are used to verify the sequence of the humanβ-crystallin A3/A1 gene promoter. The Bgl II-Sac I fragment of theresulting plasmid, pSL1180-βCrys is then excised and cloned into the BglII-Sac I sites of a CMV promoter deleted pCiNeo vector (Promega). Thiscloning is verified by restriction enzyme digestion. The resultingplasmid, pcrystiNeo, is then cleaved with Bgl II and Fsp I. Theresulting 1335 nt fragment is then cloned into the Bam HI-Hpa I sites ofpXC15-18 (FIG. 3). The resulting clone, pxcβCrys1, is verified bydigestion with numerous restriction enzymes. A restriction map of thisclone is given in FIG. 19A.

[0129] As described above for the pxcEGR clones of Example 1, four ORFs(encoding GFP, dominant negative RAS, non-phosphorylatable RB and HerpesSimplex type 1 thymidine kinase) are cloned into pxcβCrys1 to formdifferent recombinant 1 adenoviral vectors. The necessary enzymaticdigestions for the pxcβCrys clones and the sequencing of the junctionsand ORF inserts of these clones are as described above for the pxcEGRclones.

EXAMPLE 5

[0130] Construction of an Adenovirus First Stage Vector (Recombinant 1)Containing a Composite 2 Promoter with Elements from both the Rat EGR-1(Early Growth Response) Gene and Human β-crystallin Gene PromoterElements.

[0131] Plasmid pSL1180-βCrys of Example 4 is cleaved with Sca I and SacI. The resulting 305 nt fragment is isolated from an agarose gel.

[0132] Plasmid pSL1180-EGR of Example 1 is digested with Sma I and BglII, and the resulting 282 nt is isolated.

[0133] The two DNA fragments are then ligated overnight at roomtemperature. A small aliquot (1/100 of a microliter) is then used as atemplate in a PCR, using the 5′ EGR oligonucleotide of Example 1 and the3′ β-crys oligonucleotide of Example 4. The resulting 587 nt compositepromoter fragment is digested with Bgl II and Sac I, isolated from anagarose gel and ligated into the Bgl II Sac I sites of PSL 1180.Restriction enzyme digestion is used to confirm initially the cloned PCRproduct. After confirmation by sequencing of the composite promoter, theBgl II-Sac I fragment is isolated and ligated into the Bgl II-Sac Isites of Bgl II, Sac I digested pCiNeo, thus replacing the CMV promoterwith the composite promoter. This plasmid pComp2iNeo is then digestedwith Bgl II and Fsp I. The resulting 1318 nt fragment is then ligatedinto the Bam HI-Hpa I sites of pXC15-18 (FIG. 3), resulting in theplasmid pxcComp2. A restriction map of pxcComp2 is given in FIG. 20, andthe DNA sequence of its composite 2 promoter is shown in FIG. 20A.

[0134] As described above for the pxcEGR clones of Example 1, four ORFs(encoding GFP, dominant negative RAS, non-phosphorylatable RB and HerpesSimplex type 1 thymidine kinase) are cloned into pxcComp2 to formdifferent recombinant 1 adenoviral vectors. The necessary enzymaticdigestions for the pxCOMP2 clones and the sequencing of the junctionsand ORF inserts of these clones are as described above for the pxcEGRclones.

EXAMPLE 6

[0135] Evaluation of Promoter Constructs of Examples 1-5.

[0136] The proper functioning of the EGR-1 elements in the simple EGR-1promoter construct, as well as in the composite promoter constructs, istested by serum deprivation-refeeding of transiently transfected celllines. Refeeding of serum rapidly induces expression of ORFs downstreamof this promoter.

[0137] The correct functioning of MIP elements in both the simple MIPpromoter construct and in its composite promoter constructs is tested byexpression following infection of in vitro cultures of lens capsularsacs of pig eyes which have undergone cataract surgery (Liu, C. et al.,1996; Wormstone et al., 1997).

[0138] Likewise, the correct functioning of β-crystallin elements inboth the simple β-crystallin promoter construct and in its compositepromoter constructs is tested by expression following infection of invitro cultures of lens capsular sacs of pig eyes which have undergonecataract surgery.

[0139] Confirmation of the proper functioning of each portion of thecomposite promoters is achieved by analysis of GFP expressionfollowing: 1. Transient transfection of EB cells (human colon cell line)for the EGR elements and 2. recombinant adenovirus infection of in vitrocultures of lens capsular sacs of pig eyes which have undergone cataractsurgery. An intact MIP promoter construct and an intact β-crystallinpromoter construct serve as controls. As a test of GFP detection andexpression, a CMV promoter construct is also tested in parallel toassess transfection efficiency in the two cell types.

EXAMPLE 7

[0140] Creation of a Sub-line of the 293 Cell Line with Metal InducibleExpression of the Adenovirus E4 ORF 6

[0141] The 884 nt Bam HI fragment of CMV-ORF 6 obtained from GoranAkusjarvi (Uppsala, Sweden) is cloned into the Bam HI site of ametallothionein promoter vector pMT, giving rise to the clone pMT-E4ORF6 (Shaw et al., 1992) (FIG. 21). Correct orientation of the ORF 6 isevaluated with the restriction enzyme Kpn I. The MT-E4 ORF6 is thenco-transfected with the PCI-neo plasmid (Promega) into the 293 cell line(Graham et al., 1977). After three weeks of selection, G418-resistantclones are isolated and characterized for inducible ORF6 expression byNorthern analysis.

EXAMPLE 8

[0142] Recombinant Adenovirus Production, Purification andCharacterization

[0143] To produce different replication-defective recombinantadenoviruses of this invention, 293IMT-E4 ORF6 cells are plated at70-80% confluence, then co-transfected with each one of the pxc(recombinant 1) adenoviral vectors of Examples 1-5 and a modified pJM17plasmid lacking E4 ORF 6 (Bett et al., 1994), using calcium phosphate(Chen and Okayama, 1987). After 10 days, the cells are frozen and thawedthree times in 1 ml of culture media and centrifuged to remove cellulardebris. This freeze-thaw extract is then titered according to standardprocedures on 293 cells overlaid with soft agar. Individual plaques (3per desired recombinant adenovirus) are picked and eluted in 1 ml ofculture media. The individual plaques are then again titered on293/MT-E4 ORF6 cells, and the virus purification is repeated a total ofthree times.

[0144] Recombinant adenovirus stocks are then twice purified on CsCl₂gradients to remove contaminating cellular proteins. The individualtiters of recombinant adenovirus preparations are determined on 293cells as described for the isolation of individual plaques, namely onsoft agar-overlaid cultures of infected 293 cells. DNA isolated fromeach virus is analysed by diagnostic restriction enzyme mapping.

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1 13 1 704 DNA Rattus sp. EGR promoter 1 gatctagcct cagctctacgcgcctggcgc cctccctacg cgggcgtccc cgactcccgc 60 gcgcgttcag gctccgggttgggaaccaag gagggggagg gtgggtgcgc cgacccggaa 120 acaccatata aggagcaggaaggatccccc gccggaacag accttatttg ggcagcgcct 180 tatatggagt ggcccaatatggccctgccg cttccggctc tgggaggagg ggcgaacggg 240 ggttggggcg ggggcaagctgggaactcca ggagcctagc ccgggaggcc actgccgctg 300 ttccaatact aggctttccaggagcctgag cgctcagggt gccggagccg gtcgcagggt 360 ggaagcgccc accgctcttggatgggaggt cttcacgtca ctccgggtcc tcccggtcgg 420 tccttccata ttagggcttcctgcttccca tatatggcca tgtacgtcac ggcggaggcg 480 ggcccgtgct gtttcagacccttgaaatag aggccgattc ggggagtcgc gagagatccc 540 agcgcgcaga acttggggagccgccgccgc gattcgccgc cgccgccagc ttccgccgcc 600 gcaagatcgg cccctgccccagcctccgcg gcagccctgc gtccaccacg ggccgcggcc 660 accgccagcc tgggggcccacctacactcc ccgcagtgtg agct 704 2 303 DNA Homo sapiens MIP promoter 2gatctttcca gtcctgctgt tcttcacccc cacttctcgt agtctctctt gctgtgaccc 60caatcccacc ctcactgcca tggctctctc ggctcatctc ccagttgaga aaggcgggaa 120aatccagcat ttttaccatg taggggaggg gacttagccc tccacagctg tgaaggggtt 180aagaggctgg gcctgctacc tcagcctgcc cctcccaggg attaggagtc ctctataaag 240gggactgtcc acccagacaa ggccatgggg gtagcaggga cccaggcact gtgaccatga 300gct 303 3 585 DNA Artificial Sequence Description of Artificial SequenceComposite promoter 3 gatctagcct cagctctacg cgcctggcgc cctccctacgcgggcgtccc cgactcccgc 60 gcgcgttcag gctccgggtt gggaaccaag gagggggagggtgggtgcgc cgacccggaa 120 acaccatata aggagcagga aggatccccc gccggaacagaccttatttg ggcagcgcct 180 tatatggagt ggcccaatat ggccctgccg cttccggctctgggaggagg ggcgaacggg 240 ggttggggcg ggggcaagct gggaactcca ggagcctagcccgatctttc cagtcctgct 300 gttcttcacc cccacttctc gtagtctctc ttgctgtgaccccaatccca ccctcactgc 360 catggctctc tcggctcatc tcccagttga gaaaggcgggaaaatccagc atttttacca 420 tgtaggggag gggacttagc cctccacagc tgtgaaggggttaagaggct gggcctgcta 480 cctcagcctg cccctcccag ggattaggag tcctctataaaggggactgt ccacccagac 540 aaggccatgg gggtagcagg gacccaggca ctgtgaccatgagct 585 4 384 DNA Homo sapiens beta-crystallin promoter 4 gatctcccagggtcttaagg tcttaggaag atcccaaggt ggtgtgagga acntggagaa 60 ggacaagagacaagtactca tggcagagac ttctgtcctc accccctagc tgctctgaga 120 gattaagaaagccaaggcct gcagcagcca gccatgccca caacagaggg gcctctctgg 180 atttctgtatccctggttta aacaaaggcc ccagcaagct gagccaccaa agctctgggg 240 atcatgaggaacaaaggcag agggagagca gagtgctgac aggccagggc cagaggccgc 300 agggctataaagaggagggc cacagagcaa gtggtaccag atggagaccc aggctgagca 360 gcaggagctgggtgagtaag agct 384 5 591 DNA Artificial Sequence Description ofArtificial Sequence Composite promoter 5 gatctagcct cagctctacgcgcctggcgc cctccctacg cgggcgtccc cgactcccgc 60 gcgcgttcag gctccgggttgggaaccaag gagggggagg gtgggtgcgc cgacccggaa 120 acaccatata aggagcaggaaggatccccc gccggaacag accttatttg ggcagcgcct 180 tatatggagt ggcccaatatggccctgccg cttccggctc tgggaggagg ggcgaacggg 240 ggttggggcg ggggcaagctgggaactcca ggagcctagc ccactcatgg cagagacttc 300 tgtcctcacc ccctagctgctctgagagat taagaaagcc aaggcctgca gcagccagcc 360 atgcccacaa cagaggggcctctctggatt tctgtatccc tggtttaaac aaaggcccca 420 gcaagctgag ccaccaaagctctggggatc atgaggaaca aaggcagagg gagagcagag 480 tgctgacagg ccagggccagaggccgcagg gctataaaga ggagggccac agagcaagtg 540 gtaccagatg gagacccaggctgagcagca ggagctgggt gagtaagagc t 591 6 28 DNA Artificial SequenceDescription of Artificial Sequence PCR primer 6 gaagatctag cctcagctctacgcgcct 28 7 29 DNA Artificial Sequence Description of ArtificialSequence PCR primer 7 gaagagctca cactgcgggg agtgtaggt 29 8 28 DNAArtificial Sequence Description of Artificial Sequence PCR primer 8ctgaattcct tgtagaagcg cgtatggc 28 9 28 DNA Artificial SequenceDescription of Artificial Sequence PCR primer 9 cgcaagcttc tccttccgtgtttcagtt 28 10 28 DNA Artificial Sequence Description of ArtificialSequence PCR primer 10 gaagatctct tccagtcctg ctgttctt 28 11 29 DNAArtificial Sequence Description of Artificial Sequence PCR primer 11gaagagctca tggtcacagt gcctgggtc 29 12 24 DNA Artificial SequenceDescription of Artificial Sequence PCR primer 12 gaagatctcc cagggtcttaaggt 24 13 28 DNA Artificial Sequence Description of Artificial SequencePCR primer 13 gaagagctct tactcaccca gctcctgc 28

What is claimed is:
 18. (New) A replication-defective recombinant virusfor infecting lens epithelial cells of an eye, said virus comprising:(a) an open reading frame (ORF) encoding a protein that, upon expressionin a lens epithelial cell, suppresses cellular proliferation that isstimulated by eye surgery; and (b) a promoter comprising nucleotidesequences derived from at least one promoter that is active specificallyin human lens epithelial cells, wherein the promoter is functionallylinked to the ORF and expresses a biologically active amount of theprotein encoded by the ORF.
 19. (New) The replication-defectiverecombinant virus of claim 18 which is an adenovirus.
 20. (New) Thereplication-defective recombinant virus of claim 19, wherein said viruslacks E1a, E1b, and E4 ORF
 6. 21. (New) The replication-defectiverecombinant virus of claim 18 wherein the promoter sequence is one ofthe following: a) a promoter of a human Major Intrinsic Protein gene orb) a promoter of a human β A3/A1-crystallin gene or c) a compositepromoter comprising the MIP promoter or the β-crystallin promoter, incombination with elements of an early growth response gene promoter. 22.(New) The replication-defective recombinant virus of claim 21 whereinthe promoter sequence is one of the following: a) a promoter comprisingthe portion of the human Major Intrinsic Protein gene from −259 nt to+34 nt of SEQ ID NO:2, or b) a promoter comprising the portion of thehuman β A3/A1-crystallin gene from −345 nt to +45 nt of SEQ ID NO:4, orc) a composite promoter comprising i) at least one element chosen fromthe group consisting of: the promoter of the human Major IntrinsicProtein gene, the portion of the promoter of the human Major IntrinsicProtein gene from −259 nt to −34 nt of SEQ ID NO:2, the β-crystallinpromoter, and the portion of the human β-crystallin gene from −345 nt to+45 nt of SEQ ID NO:4; in combination with ii) the portion of thepromoter of the rat Early Growth Response-1 gene from −518 nt to −236 ntof SEQ ID NO:1.
 23. (New) The replication-defective recombinant virus ofclaim 18, wherein the promoter sequence is non-methylated.
 24. (New) Amethod for the treatment of an eye of a patient, undergoing eye surgery,in order to reduce the incidence of cellular proliferation in the eyefollowing the eye surgery, and thereby prevent the formation ofsecondary cataracts, comprising the step of: treating the eye with areplication-defective recombinant virus of claim
 18. 25. (New) Themethod of claim 24 wherein the eye is treated during the eye surgerywith the recombinant virus.
 26. (New) The method of claim 25, comprisingthe additional step of treating the eye at least one more time with therecombinant virus after the surgery.
 27. (New) The method of claim 24,comprising the additional step of treating the patient with acyclovir ora nucleoside analogue structurally related to acyclovir when therecombinant virus contains a thymidine kinase ORF of a Herpes virus orthe UL97 ORF of a Cytomegalovirus.
 28. (New) A composition for thetreatment of an eye, undergoing eye surgery, in order to reduce theincidence of cellular proliferation of the lens epithelial cells in theeye following surgery and thereby prevent secondary cataracts,comprising the replication-defective recombinant virus of claim
 18. 29.(New) A replication-defective recombinant adenovirus virus comprising anORF selected from the group consisting of: a) a non-phosphorylatableretinoblastoma ORF; b) a dominant negative mutant of a RAS ORF; or c) athymidine kinase ORF of a Herpes virus; wherein i) said ORF is under thecontrol of a promoter sequence comprising nucleotide sequences derivedfrom at least one promoter that is active specifically in human lensepithelial cells; ii) said replication-defective recombinant virus iscapable of infecting lens epithelial cells of an open lens capsule of aneye.
 30. (New) The replication-defective recombinant adenovirus of claim29 which lacks E1a, E1b and E4 ORF
 6. 31. (New) Thereplication-defective recombinant virus of claim 29 wherein the promotersequence is one of the following: a) the promoter of a human MajorIntrinsic Protein gene in SEQ ID NO:2; or b) the promoter of a human βA3/A1-crystallin gene in SEQ ID NO:4.
 32. (New) Thereplication-defective recombinant adenovirus of claim 31 which lacksE1a, E1b and E4 ORF
 6. 33. (New) A method for the treatment of an eye ofa patient, undergoing eye surgery, in order to reduce the incidence ofcellular proliferation in the eye following the eye surgery, and therebyprevent the formation of secondary cataracts, comprising the step oftreating the eye with a replication-defective recombinant adenovirusvirus of claim
 29. 34. (New) A method for the treatment of an eye of apatient, undergoing eye surgery, in order to reduce the incidence ofcellular proliferation in the eye following the eye surgery, and therebyprevent the formation of secondary cataracts, comprising the step oftreating the eye with a replication-defective recombinant adenovirusvirus of claim
 32. 35. (New) The replication-defective recombinant virusof claim 18, wherein the promoter sequence is a composite promotersequence comprising a basal transcription region and at least onetranscription enhancer element, the enhancer including but not limitedto an immediate early enhancer.
 36. (New) The replication-defectiverecombinant virus of claim 29, wherein the promoter sequence is acomposite promoter sequence comprising a basal transcription region andat least one transcription enhancer element, the enhancer including butnot limited to an immediate early enhancer.